Isolation, detection, diagnosis and/or characterization of circulating Trop-2-positive cancer cells

ABSTRACT

Described herein are compositions and methods of use of anti-Trop-2 antibodies or antigen-binding fragment thereof to isolate, enrich, detect, diagnose and/or characterize circulating tumor cells (CTCs) from patients with a Trop-2 positive cancer. Preferably, the antibody is an RS7, 162-46.2 or MAB650 antibody. The compositions and methods are of use to detect, diagnose and/or treat metastatic Trop-2 +  cancers, such as breast, ovarian, cervical, endometrial, lung, prostate, colon, rectum, stomach, esophageal, bladder, renal, pancreatic, thyroid, epithelial or head-and-neck cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/135,758, filed Apr. 22, 2016, which claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application 62/151,169, filedApr. 22, 2015, the text of which is incorporated herein by reference inits entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 21, 2016, isnamed IMM359US1 SL.txt and is 44,983 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to methods and compositions for isolating,detecting, diagnosing and/or characterizing Trop-2+ cancer cells,preferably from the circulation. The methods and compositions utilizeanti-Trop-2 antibodies, which may be monovalent, bivalent ormultivalent. In a preferred embodiment, anti-Trop-2 antibodies are thesole anti-TAA (tumor-associated antigen) capture antibodies utilized inthe assay, which does not include use of mixtures of antibodies againstTAAs other than Trop-2. In alternative embodiments, the capture antibodymay be a bispecific antibody comprising an anti-Trop-2 antibody orfragment and a second antibody or fragment against a different TAA. Morepreferably, the antibodies are rodent, chimeric, humanized or humanantibodies or antigen-binding fragments thereof. Expression of Trop-2 incancer cells may be assessed using known techniques, including but notlimited to binding of anti-Trop-2 antibodies as detected by flowcytometry or immunohistochemistry, and quantitative RT-PCR. Automatedsystems and devices that have been developed to isolate and/or detectcirculating tumor cells (CTCs), including but not limited to theMagSweeper device (Illumina, Inc., San Diego, Calif.), LIQUIDBIOPSY®system (Cynvenio Biosystems, Inc., Westlake Village, Calif.),CELLSEARCH® system (Vendex LLC, Raritan, N.J.), GILUPI CELLCOLLECTOR™(GILUPI GmbH, Potsdam, Germany), APOSTREAM® system (Apocell, Houston,Tex.), ONCOCEE™ microfluidic platform (BioCept Laboratories, San Diego,Calif.), VerIFAST System (Casavant et al., 2013, Lab Chip 13:391-6;2014, Lab Chip 14:99-105) or ISOFLUX™ system (Fluxion, South SanFrancisco, Calif.) may be utilized in the practice of the claimedmethods. Most preferably, the anti-Trop-2 antibody is a murine, chimericor humanized RS7 (hRS7) antibody, comprising the light chain CDRsequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2);and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO:6). However, in alternative embodiments otherknown anti-Trop-2 antibodies may be utilized, as discussed below. Themethods and compositions are applicable for the enrichment, isolation,detection, diagnosis and/or characterization of various metastaticTrop-2-expressing cancers, such as breast (e.g., triple-negative breastcancer), ovarian, cervical, endometrial, lung, prostate, colon, rectum,stomach, esophageal, bladder, renal, pancreatic, thyroid, epithelial,and head-and-neck cancers. Anti-Trop-2 antibodies may be utilized incombination with one or more labeled detection antibodies, or may bedirectly labeled by conjugation with at least one diagnostic agent.Alternatively, a bispecific antibody may comprise one binding site forTrop-2 and another binding site for a hapten on a targetable construct,typically a small peptide labeled with at least one diagnostic agent. Incertain alternative embodiments, detection of Trop-2⁺ CTCs may befollowed by therapeutic treatment of the Trop-2⁺ cancer, usinganti-Trop-2 antibodies or fragments thereof. Preferably the antibody orfragment is conjugated to at least one therapeutic agent, such asantibodies, antibody fragments, drugs, toxins, nucleases, hormones,immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boroncompounds, photoactive agents or dyes or radioisotopes. More preferably,the therapeutic agent is SN-38 or P2PDOX.

Related Art

Trop-2 (human trophoblast-cell-surface marker) is a cell surfaceglycoprotein that was originally identified in normal and malignanttrophoblast cells (Lipinski et al., 1981, Proc Natl. Acad Sci USA78:5147-50). Trop-2 is highly expressed in most human carcinomas,particularly in epithelial carcinomas and adenocarcinomas, with reportedlow to restricted expression in normal tissues (see, e.g., Cubas et al.,2010, Molec Cancer 9:253; Stepan et al., 2011, J Histochem Cytochem59:701-10; Varughese et al., 2011, Am J Obst Gyn 205:567e-e7).Expression of Trop-2 is associated with metastasis, increased tumoraggressiveness and decreased patient survival (Cubas et al., 2010;Varughese et al., 2011). Pathogenic effects of Trop-2 have been reportedto be mediated, at least in part, by the ERK 1/2 MAPK pathway (Cubas etal., 2010).

It has been proposed that early in tumor progression, cancer cells maybe found in low concentration in the circulation (see, e.g.,Krishnamurthy et al., 2013, Cancer Medicine 2:226-33; Alix-Panabieres &Pantel, 2013, Clin Chem 50:110-18; Wang et al., Feb. 24, 2015, Int JClin Oncol, Epub ahead of print). Due to the relatively non-invasivenature of blood sample collection, there has been great interest in theisolation and detection of CTCs, to promote cancer diagnosis at anearlier stage of the disease and as a predictor for tumor progression,disease prognosis and/or responsiveness to drug therapy (see, e.g.,Alix-Panabieres & Pantel, 2013, Clin Chem 50:110-18; Winer-Jones et al.,2014, PLoS One 9:e86717; U.S. Patent Appl. Publ. No. 2014/0357659).

Various techniques and apparatus have been developed to isolate and/ordetect circulating tumor cells. Several reviews of the field haverecently been published (see, e.g., Alix-Panabieres & Pantel, 2013, ClinChem 50:110-18; Joosse et al., 2014, EMBO Mol Med 7:1-11; Truini et al.,2014, Fron Oncol 4:242). The techniques have involved enrichment and/orisolation of CTCs, generally using capture antibodies against an antigenexpressed on tumor cells, and separation with magnetic nanoparticles,microfluidic devices, filtration, magnetic separation, centrifugation,flow cytometry and/or cell sorting devices (e.g., Krishnamurthy et al.,2013, Cancer Medicine 2:226-33; Alix-Panabieres & Pantel, 2013, ClinChem 50:110-18; Joosse et al., 2014, EMBO Mol Med 7:1-11; Truini et al.,2014, Fron Oncol 4:242; Powell et al., 2012, PLoS ONE 7:e33788;Winer-Jones et al., 2014, PLoS One 9:e86717; Gupta et al., 2012,Biomicrofluidics 6:24133; Saucedo-Zeni et al., 2012, Int J Oncol41:1241-50; Harb et al., 2013, Transl Oncol 6:528-38). The enriched orisolated CTCs may then be analyzed using a variety of known methods, asdiscussed further below. Systems or apparatus that have been used forCTC isolation and detection include the CELLSEARCH® system (e.g., Truiniet al., 2014, Front Oncol 4:242), MagSweeper device (e.g., Powell etal., 2012, PLoS ONE 7:e33788), LIQUIDBIOPSY® system (Winer-Jones et al.,2014, PLoS One 9:e86717), APOSTREAM® system (e.g., Gupta et al., 2012,Biomicrofluidics 6:24133), GILUPI CELLCOLLECTOR™ (e.g., Saucedo-Zeni etal., 2012, Int J Oncol 41:1241-50), and ISOFLUX™ system (Harb et al.,2013, Transl Oncol 6:528-38).

To date, the only FDA-approved technology for CTC detection involves theCELLSEARCH® platform (Vendex LLC, Raritan, N.J.), which utilizesanti-EpCAM antibodies attached to magnetic nanoparticles to captureCTCs. Detection of bound cells occurs with fluorescent-labeledantibodies against cytokeratin (CK) and CD45. Fluorescently labeledcells bound to magnetic particles are separated out using a strongmagnetic field and are counted by digital fluorescence microscopy. TheCELLSEARCH® system has received FDA approval for detection of metastaticbreast, prostate and colorectal cancers.

Most CTC detection systems have focused on use of anti-EpCAM captureantibodies (see, e.g., Truini et al., 2014, Front Oncol 4:242; Powell etal., 2012, PLoS ONE 7:e33788; Alix-Panabieres & Pantel, 2013, Clin Chem50:110-18; Lin et al., 2013, Biosens Bioelectron 40:63-67; Wang et al.,Feb. 24, 2015, Int J Clin Oncol Epub ahead of print; Magbanua et al.,2015, Clin Cancer Res 21:1098-105; Harb et al., 2013, Transl Oncol6:528-38). However, not all metastatic tumors express EpCAM (see, e.g.,Mikolajcyzyk et al., 2011, J Oncol 2011:252361; Pecot et al., 2011,Cancer Discovery 1:580-86; Gupta et al., 2012, Biomicrofluidics6:24133). Attempts have been made to utilize alternative schemes forisolating and detecting EpCAM-negative CTCs, such as use of antibodycombinations against TAAs. Antibodies against as many as 10 differentTAAs have been utilized in an attempt to increase recovery of metastaticcirculating tumor cells (e.g., Mikolajcyzyk et al., 2011, J Oncol2011:252361; Pecot et al., 2011, Cancer Discovery 1:580-86;Krishnamurthy et al., 2013, Cancer Medicine 2:226-33; Winer-Jones etal., 2014, PLoS One 9:e86717).

Drawbacks exist to such approaches, including the complexity ofpreparing and using large numbers of different antibodies and theirattachment to magnetic nanoparticles, microfluidic devices or otherseparation technologies, as well as potential cross-reactivity againstnormal cell populations when using a broad spectrum of anti-tumorantibodies. A need exists in the art for improved methods of isolating,detecting, diagnosing and/or characterizing CTCs, using antibodiesagainst a single TAA that is expressed in a broad range of tumors.

SUMMARY

In various embodiments, the present invention concerns enrichment,isolation, detection, diagnosis and/or characterization ofTrop-2-positive circulating tumor cells (CTCs) using anti-Trop-2antibodies and/or antigen-binding fragments thereof. The anti-Trop-2antibody may be used to enrich and/or isolate tumor cells from thecirculation. Bound CTCs may be detected by a variety of known techniquesand/or apparatus, as discussed in detail below. Any known method fordetecting biomarkers of isolated CTCs may be utilized, such as FISH,FACS, fluorescence microscopy, fluorescent detection, flow cytometry,immunohistochemistry, microchip-based systems, RT-PCR, ELISA, or anyother technique known in the art for detecting the presence of cancercells.

In a specific embodiment, the anti-Trop-2 antibody may be a murine,chimeric or humanized RS7 antibody (see, e.g., U.S. Pat. No. 7,238,785,the Figures and Examples section of which are incorporated herein byreference), comprising the light chain CDR sequences CDR1 (KASQDVSIAVA,SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ IDNO:3) and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).However, as discussed below other anti-Trop-2 antibodies are known andmay be used.

The anti-Trop-2 antibody moiety may be a monoclonal antibody, anantigen-binding antibody fragment, a bispecific or multivalent antibody,or other antibody-based molecule. The antibody can be of variousisotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferablycomprising human IgG1 hinge and constant region sequences. The antibodyor fragment thereof can be a rodent, chimeric, a humanized, or a humanantibody, as well as variations thereof, such as half-IgG4 antibodies(referred to as “unibodies”), as described by van der Neut Kolfschotenet al. (Science 2007; 317:1554-1557). More preferably, the antibody orfragment thereof may be designed or selected to comprise human constantregion sequences that belong to specific allotypes, such as G1m3,G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selectedfrom the group consisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Where bispecific antibodies are used to capture CTCs, the antibody maycomprises at least one anti-Trop-2 antibody or fragment thereof, and atleast one antibody or fragment thereof against a different TAA.Exemplary TAAs may include carbonic anhydrase IX, CCL19, CCL21, CSAp,CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37,CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e,CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147,CD154, CXCR4, CXCR7, CXCL12, HIF-1α, AFP, PSMA, CEACAM5, CEACAM-6,c-met, B7, ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor,GROB, HMGB-1, hypoxia inducible factor (HIF), insulin-like growthfactor-1 (ILGF-1), IFN-γ, IFN-α, IL-β, IL-2, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4,MUC5ac, NCA-95, NCA-90, Ia, EGP-1, EGP-2, HLA-DR, tenascin, Le(y),RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens, tumornecrosis antigens, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF,complement factors C3, C3a, C3b, C5a, or C5. Preferably, the TAA isselected from the group consisting of CEACAM5, MUC5ac, CD74, HLA-DR,CSAp, AFP (alpha-fetoprotein), HER2, vimentin, EGFR, IGF-1R, PD-L1 andPD-L2.

Because the detected tumors will be Trop-2-positive, they may be treatedwith anti-Trop-2 antibodies, such as anti-Trop-2 antibody-drugconjugates (ADCs). An anti-Trop-2 antibody may initially be used todetect and/or quantify expression or gene copy number of Trop-2 in theCTC. Such analysis may be used to predict response to therapeuticanti-Trop-2 antibodies, as well as to monitor response of the tumor(s)to treatment. As discussed below, immunoconjugates of anti-Trop-2antibodies may include any known therapeutic agent, such as achemotherapeutic agent. A number of cytotoxic drugs of use for cancertreatment are well-known in the art and any such known drug may beconjugated to the antibody of interest. In a preferred embodiment, thedrug conjugated to the antibody is a camptothecin or anthracycline, mostpreferably SN-38 or a pro-drug form of 2-pyrrolinodoxorubicin (2-PDox)(see, e.g., U.S. Pat. Nos. 8,877,202 and 8,750,496, the Figures andExamples section of each incorporated herein by reference). The drug tobe conjugated to the anti-Trop-2 antibody or antibody fragment may beselected from the group consisting of an anthracycline, a camptothecin,a tubulin inhibitor, a maytansinoid, a calicheamycin, an auristatin, anitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, anitrosourea, a triazene, a folic acid analog, a taxane, a COX-2inhibitor, a pyrimidine analog, a purine analog, an antibiotic, anenzyme inhibitor, an epipodophyllotoxin, a platinum coordinationcomplex, a vinca alkaloid, a substituted urea, a methyl hydrazinederivative, an adrenocortical suppressant, a hormone antagonist, anantimetabolite, an alkylating agent, an antimitotic, an anti-angiogenicagent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shockprotein (HSP90) inhibitor, a proteosome inhibitor, an HDAC inhibitor, apro-apoptotic agent, and a combination thereof.

The anti-Trop-2 antibodies are of use for detection, diagnosis,characterization and/or treatment of Trop-2 expressing cancers, such asbreast, ovarian, cervical, endometrial, lung, prostate, colon, rectum,stomach, esophageal, bladder, renal, pancreatic, thyroid, epithelial orhead-and-neck cancers. The methods and compositions may be of particularuse for detection and/or treatment of metastatic colorectal cancer,triple-negative breast cancer, HER+, ER+, progesterone+ breast cancer,metastatic non-small-cell lung cancer (NSCLC), metastatic small-celllung cancer (SCLC), metastatic pancreatic cancer, metastatic renal cellcarcinoma, metastatic gastric cancer, metastatic esophageal cancer,metastatic urothelial cancer, or metastatic prostate cancer.

DETAILED DESCRIPTION

Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, sFv and the like. Antibody fragments may also include singledomain antibodies and IgG4 half-molecules, as discussed below.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the full-length antibody. The term“antibody fragment” also includes isolated fragments consisting of thevariable regions of antibodies, such as the “Fv” fragments consisting ofthe variable regions of the heavy and light chains and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (“scFv proteins”).

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples section of which are incorporated herein by reference.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes, contrast agents, luminescent agents,chemiluminescent agents, fluorescent compounds or molecules andenhancing agents (e.g., paramagnetic ions). Preferably, the diagnosticagents are selected from the group consisting of radioisotopes,enhancing agents, and fluorescent compounds.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptoticagents, anti-angiogenic agents, boron compounds, photoactive agents ordyes and radioisotopes. Therapeutic agents of use are described in moredetail below.

An immunoconjugate is an antibody, antibody fragment or fusion proteinconjugated to at least one therapeutic and/or diagnostic agent.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. Multispecific, multivalentantibodies are constructs that have more than one binding site, and thebinding sites are of different specificity.

A bispecific antibody is an antibody that can bind simultaneously to twodifferent targets. Bispecific antibodies (bsAb) and bispecific antibodyfragments (bsFab) may have at least one arm that specifically binds to,for example, a tumor-associated antigen and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering.

FIGURE LEGENDS

FIG. 1. Analysis of Trop-2 copy number by FISH. MCF-7 (Trop-2 positive)cells were analyzed by FISH. Trop-2 copy number was determined usinganti-Trop-2 and anti-chromosome-1 specific probes (Empire Genomics,Buffalo, N.Y.).

FIG. 2. Analysis of Trop-2 copy number by FISH. A549 (Trop-2 negative)cells were analyzed by FISH. Trop-2 copy number was determined usinganti-Trop-2 and anti-chromosome-1 specific probes (Empire Genomics,Buffalo, N.Y.).

FIG. 3. Analysis of topoisomerase-I copy number by FISH. MCF-7 cellswere analyzed by FISH. Topoisomerase I (TOP1) copy number was determinedusing anti-TOP1 and anti-chromosome-20 specific probes (ABNOVA®, Taipei,Taiwan).

FIG. 4. Analysis of topoisomerase-I copy number by FISH. A549 cells wereanalyzed by FISH. Topoisomerase I (TOP1) copy number was determinedusing anti-TOP1 and anti-chromosome-20 specific probes (ABNOVA®, Taipei,Taiwan).

ANTI-TROP-2 ANTIBODIES

The subject methods and compositions for CTC isolation and/or detectionutilize at least one antibody or fragment thereof that binds to Trop-2,including rodent, chimeric, human or humanized antibodies. In a specificpreferred embodiment, the anti-Trop-2 antibody may be a humanized RS7antibody (see, e.g., U.S. Pat. No. 7,238,785, incorporated herein byreference in its entirety), comprising the light chain CDR sequencesCDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3(QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (NYGMN,SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3(GGFGSSYWYFDV, SEQ ID NO:6).

The RS7 antibody was a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its roleas a tumor-associated calcium signal transducer (Ripani et al., Int JCancer 1998; 76:671-6), the expression of human Trop-2 was shown to benecessary for tumorigenesis and invasiveness of colon cancer cells,which could be effectively reduced with a polyclonal antibody againstthe extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5). Trop-2 is highly expressed in the vast majority of humantumors and animal models of cancer (McDougall et al., 2015, Dev Dyn244:99-109).

The utility of Trop-2 as a marker for solid cancers (Cubas et al.,Biochim Biophys Acta 2009; 1796:309-14) is attested by further reportsthat documented the clinical significance of overexpressed Trop-2 inbreast (Huang et al., Clin Cancer Res 2005; 11:4357-64), colorectal(Ohmachi et al., Clin Cancer Res 2006; 12:3057-63; Fang et al., Int JColorectal Dis 2009; 24:875-84), and oral squamous cell (Fong et al.,Modern Pathol 2008; 21:186-91) carcinomas. The latest evidence thatprostate basal cells expressing high levels of Trop-2 are enriched forin vitro and in vivo stem-like activity is particularly noteworthy(Goldstein et al., Proc Natl Acad Sci USA 2008; 105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue (Stein et al., 1990). Trop-2 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991). Drug-conjugated RS7 MAb in animal models alsohave shown targeting and therapeutic efficacy of human cancer xenografts(Cardillo et al., Clinical Cancer Res., 17:3157-69, 2011).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Stein et al., Int. J.Cancer 55:938, 1993) Both cell types stained strongly, indicating thatthe RS7 antibody does not distinguish between histologic classes ofnon-small-cell carcinoma of the lung.

While the hRS7 antibody is preferred, other anti-Trop-2 antibodies areknown and/or publicly available and in alternative embodiments may beutilized in the subject methods and compositions. While humanized orhuman antibodies are preferred for reduced immunogenicity, inalternative embodiments a chimeric antibody may be of use, while rodentMAbs can be useful for in-vitro and ex-vivo studies. As discussed below,methods of antibody humanization are well known in the art and may beutilized to convert an available murine or chimeric antibody into ahumanized form.

Anti-Trop-2 antibodies are commercially available from a number ofsources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417(LifeSpan BioSciences, Inc., Seattle, Wash.); 10428-MM01, 10428-MM02,10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54(eBioscience, San Diego, Calif.); sc-376181, sc-376746, Santa CruzBiotechnology (Santa Cruz, Calif.); MM0588-49D6, (Novus Biologicals,Littleton, Colo.); ab79976, and ab89928 (ABCAM®, Cambridge, Mass.).

Other anti-Trop-2 antibodies have been disclosed in the patentliterature. For example, U.S. Publ. No. 2013/0089872 disclosesanti-Trop-2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107(Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253),T6-16 (Accession No. FERM BP-11346), and T5-86 (Accession No. FERMBP-11254), deposited with the International Patent Organism Depositary,Tsukuba, Japan. U.S. Pat. No. 5,840,854 disclosed the anti-Trop-2monoclonal antibody BR110 (ATCC No. HB11698). U.S. Pat. No. 7,420,040disclosed an anti-Trop-2 antibody produced by hybridoma cell lineAR47A6.4.2, deposited with the IDAC (International Depository Authorityof Canada, Winnipeg, Canada) as accession number 141205-05. U.S. Pat.No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridomacell line AR52A301.5, deposited with the IDAC as accession number141205-03. U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representativeantibody were deposited with the American Type Culture Collection(ATCC), Accession Nos. PTA-12871 and PTA-12872. U.S. Pat. No. 8,715,662discloses anti-Trop-2 antibodies produced by hybridomas deposited at theAID-ICLC (Genoa, Italy) with deposit numbers PD 08019, PD 08020 and PD08021. U.S. Patent Application Publ. No. 20120237518 disclosesanti-Trop-2 antibodies 77220, KM4097 and KM4590. U.S. Pat. No. 8,309,094(Wyeth) discloses antibodies A1 and A3, identified by sequence listing.The Examples section of each patent or patent application cited above inthis paragraph is incorporated herein by reference. For non-patentpublications, Lipinski et al. (1981, Proc Natl. Acad Sci USA,78:5147-50) disclosed anti-Trop-2 antibodies 162-25.3 and 162-46.2. Morerecently, the Pr1E11 anti-Trop-2 antibody was reported to recognize aunique epitope on Trop-2 (Ikeda et al., Biochem Biophys Res Comm458:877-82).

Numerous anti-Trop-2 antibodies are known in the art and/or publiclyavailable. As discussed below, methods for preparing antibodies againstknown antigens were routine in the art. The sequence of the human Trop-2protein was also known in the art (see, e.g., GenBank Accession No.CAA54801.1). Methods for producing humanized, human or chimericantibodies were also known. The person of ordinary skill, reading theinstant disclosure in light of general knowledge in the art, would havebeen able to make and use the genus of anti-Trop-2 antibodies.

None of the prior studies discussed above contained any disclosure ofthe use of anti-Trop-2 antibodies for isolating or detecting Trop-2positive CTCs. A need exists for compositions and methods for enriching,isolating, detecting, diagnosing and/or characterizing Trop-2 positiveCTCs.

Isolation and Detection of Circulating Tumor Cells

The anti-Trop-2 antibodies may be utilized to enrich, isolate, detectand/or diagnose Trop-2 positive CTCs using any known technology for CTCisolation and detection. Numerous systems have been developed and arecommercially available for CTC detection. Although the majority weredeveloped using specific anti-EpCAM antibodies, the compositions andmethods may be modified to utilize anti-Trop-2 antibodies instead. Thus,isolation and detection of Trop-2 positive CTCs may be performed usingany such known system, or more traditional methods of cell isolation anddetection. Non-limiting examples of such known techniques are discussedbelow.

The present invention may be used with an affinity-based enrichmentstep, as well as methods without an enrichment steps, such as MAINTRAC®(Pachmann et al. 2005, Breast Cancer Res, 7: R975). Methods that use amagnetic device for affinity-based enrichment, include the CELLSEARCH®system (Veridex), the LIQUIDBIOPSY® platform (Cynvenio Biosystems) andthe MagSweeper device (Talasaz et al, PNAS, 2009, 106: 3970). Methodsthat do not use a magnetic device for affinity-based enrichment, includea variety of fabricated microfluidic devices, such as CTC-chips (Stottet al. 2010, Sci Transl Med, 2: 25ra23), EIB-chips (Stott et al, 2010,PNAS, 107: 18392), NanoVelcro chips (Lu et al., 2013, Methods, 64: 144),GEDI microdevice (Kirby et al., 2012, PLoS ONE, 7: e35976), andBiocept's ONCOCEE™ technology (Pecot et al., 2011, Cancer Discov, 1:580).

Use of the FDA-approved CELLSEARCH® system for CTC detection innon-small cell and small cell lung cancer patients is discussed inTruini et al. (2014, Front Oncol 4:242). A 7.5 ml sample of peripheralblood is mixed with magnetic iron nanoparticles coated with annanti-EpCAM antibody. A strong magnetic field is used to separate EpCAMpositive from EpCAM-negative cells. Detection of bound CTCs wasperformed using fluorescently labeled anti-CK and anti-CD45 antibodies,along with DAPI (4′,6′diamidino-2-phynlindole) fluorescent labeling ofcell nuclei. CTCs were identified by fluorescent detection as CKpositive, CD45 negative and DAPI positive.

The VerIFAST system was used for diagnosis and pharmacodynamic analysisof circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC)(Casavant et al., 2013, Lab Chip 13:391-6; 2014, Lab Chip 14:99-105).The VerIFAST platform utilizes the relative dominance of surface tensionover gravity in the microscale to load immiscible phases side by side.This pins aqueous and oil fields in adjacent chambers to create avirtual filter between two aqueous wells (Casavant et al., 2013, LabChip 13:391-6). Using paramagnetic particles (PMPs) with attachedantibody or other targeting moieties, specific cell populations can betargeted and isolated from complex backgrounds through a simple traverseof the oil barrier. In the NSCLC example, streptavidin was conjugated toDYNABEADS® FLOWCOMP™ PMPs (Life Technologies, USA) and cells werecaptured using biotinylated anti-EpCAM antibody. A handheld magnet wasused to transfer CTCs bound to PMPs between aqueous chambers. CollectedCTCs were released with PMP release buffer (DYNABEADS®) and stained forEpCAM, EGFR or transcription termination factor (TTF-1).

The VerIFAST platform integrates a microporous membrane into an aqueouschamber to enable multiple fluid transfers without the need for celltransfer or centrifugation. With physical characteristic scales enablinghigh precision relative to macroscale techniques, such microfluidictechniques are well adapted to capture and assess CTCs with minimalsample loss. The VerIFAST platform effectively captured CTCs from bloodof NSCLC patients.

The GILUPI CELLCOLLECTOR™ (Saucedo-Zeni et al., 2012, Int J Oncol41:1241-50) is based on a functionalized medical Seldinger guidewire(FSMW) coated with chimeric anti-EpCAM antibody. The guidewire wasfunctionalized with a polycarboxylate hydrogel layer that was activatedwith EDC and NHS, allowing covalent bonding of antibody. Theantibody-coated FSMW was inserted in the cubital veins of breast canceror NSCLC lung cancer patients through a standard venous cannula for 30minutes. Following binding of cells to the guidewire, CTCs wereidentified by immunocytochemical staining of EpCAM and/or cytokeratinsand nuclear staining. Fluorescent labeling was analyzed with an AxioImager.Alm microscope (Zeiss, Jena, Germany) equipped with an AxioCamdigital camera system and AxioVision 4.6 software. The FSMW system wascapable of enriching EpCAM-positive CTCs from 22 of 24 patients tested,including those with early stage cancer in which distant metasteses hadnot yet been diagnosed. No CTCs were detected in healthy volunteers. Anadvantage of the FSMW system is that it is not limited by the volume ofex vivo blood samples that may be processed using alternativemethodologies, such as the CELLSEARCH® system. Estimated blood volume incontact with the FSMW during the 30 minute exposure was 1.5 to 3 liters.

The MagSweeper device (e.g., Powell et al., 2012, PLoS ONE 7:e33788) isanother system utilized antibody-coated magnetic particles for CTCdetection. Nine milliliters of whole blood was mixed ex vivo for 1 hr atRT with 4.5 μM DYNABEADS® (Invitrogen, Life Technologies, Grand Island,N.Y.) coated with the BerEP4 anti-EpCAM antibody. After dilution withPBS, cells bound to DYNABEADS® were captured by a sweeping magneticdevice (MagSweeper, see FIG. 1 of Powel et al., 2012). Two cycles ofcapture-wash-relase were performed, using a controlled shear force thatreleased non-specifically bound leukocytes and RBCs. Captured cells werereleased into fresh buffer and examined using an Axio Observer A1inverted microscope (Zeiss). Single CTCs were manually aspirated andstored frozen, prior to analysis of expression of 87 genes by chip basedhigh-throughput qRT-PCR.

Gupta et al. (2012, Biomicrofluidics 6:24133) discussed use of theAPOSTREAM™ dielectrophoretic device for CTC collection and analysis. Amicrofluidic flow chamber is used with dielectrophoretic (DEP)technology to capture CTCs (see FIG. 1, Gupta et al., 2012). The systemmay be operated in continuous mode for flow-through isolation andenrichment of CTCs from peripheral blood. DEP sorts cells with distinctbiophysical characteristics by exploiting the frequency-dependentdielectric properties of different cell types, arising from differencesin morphologic properties and electrical conductivity. These differencesresult in differential frequency-dependent migration of CTCs and normalcells in the microfluidic chamber. At an AC frequency in the range of45-85 kHz, cancer cells experience a positive (attractive) DEP force,which causes them to migrate towards the electrode plane and away fromthe hydrodynamic flow through the chamber. At the same frequency, normalcells experience a negative (repulsive) DEP force, which moves them intothe hydrodynamic flow velocity profile and out of the chamber. Acollection port is used to remove separated CTCs for further analysis.For the initial optimization study, cultured cancer cells were spikedinto normal blood mononuclear cells and were recovered with over a 70%efficiency. Although the APOSTREAM™ system disclosed by Gupta does notuse capture antibodies, the subject anti-Trop-2 antibodies maypotentially be utilized to increase the efficiency of CTC separationand/or for post-separation characterization of the isolated CTCs.

Winer-Jones et al. (2014, PLoS One 9:e86717) discussed use of theLIQUIDBIOPSY® system for isolation and characterization of CTCs. TheLIQUIDBIOPSY® system uses high throughput sheath flow microfluidicsthrough a flow cell, combined with anti-EpCAM antibodies as a captureagent. Biotinylated anti-EpCAM was attached to streptavidin-coated IMAG™beads (BD, Franklin Lakes, N.J.) and mixed with blood samples,containing spiked tumor cells labeled with CFSE or FITC. Normalnucleated cells were labeled with DAPI. After antibody binding, theblood samples were processed on the CTC flow cell, attached to a glassslide. An external magnetic field is used to capture magnetic-bead boundCTCs on the glass surface, separating them from the laminar flowcontaining normal cells. Captured cells were counted using an EclipseE80i fluorescent microscope (Nikon Instruments, Melville, N.Y.).

The person of ordinary skill will realize that any of these systems, orany other known system for CTC enrichment and/or isolation, may be usedwith the subject anti-Trop-2 antibodies for enrichment, isolation,detection and/or characterization of CTCs. Where an anti-Trop-2 captureantibody is utilized, the bound CTCs may be detected and/orcharacterized using labeled antibodies against a different Trop-2epitope, or against other known tumor-associated antigens, including butnot limited to carbonic anhydrase IX, CCL19, CCL21, CSAp, CD1, CD1a,CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R,CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70,CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4,CXCR7, CXCL12, HIF-1-α, AFP, PSMA, CEACAM5, CEACAM-6, c-met, B7, ED-B offibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1,hypoxia inducible factor (HIF), insulin-like growth factor-1 (ILGF-1),IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R,IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1,MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, NCA-95, NCA-90, Ia,EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAILreceptor (R1 and R2), VEGFR, EGFR, PlGF, complement factors C3, C3a,C3b, C5a, and C5.

Antibody Preparation

Techniques for preparing monoclonal antibodies against virtually anytarget antigen, such as Trop-2, are well known in the art. See, forexample, Köhler and Milstein, Nature 256: 495 (1975), and Coligan et al.(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (JohnWiley & Sons 1991). Briefly, monoclonal antibodies can be obtained byinjecting mice with a composition comprising an antigen, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce antibodies to the antigen, culturing the clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Where antibodies are to be utilized in vivo,for example in tumor therapy following detection of a Trop-2 positivecancer, such fully human antibodies are expected to exhibit even fewerside effects than chimeric or humanized antibodies and to function invivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, 1 Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies and Target Antigens

As discussed above, in certain alternative embodiments the anti-Trop-2antibodies are of use for treating Trop-2-expressing cancers, followingdetection of circulating Trop-2-positive tumor cells. In someembodiments, the target cancer may express one or more additionaltumor-associated antigens (TAAs) that may be targeted for tumor therapy.Particular antibodies that may be of use for therapy of cancer include,but are not limited to, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22),veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab(GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor), nivolumab(anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-epithelialglycoprotein-1 (EGP-1, also known as Trop-2)), PAM4 or KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);PAM4 (aka clivatuzumab, anti-mucin) and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20050271671;20060193865; 20060210475; 20070087001; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMIVIU-31 (U.S. Pat. No. 7,300,655),hLL1 (U.S. Pat. No. 7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9(U.S. Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S.Pat. No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4),abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab(anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101,anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), and Benlysta (HumanGenome Sciences).

Other useful tumor-associated antigens that may be targeted includecarbonic anhydrase IX, B7, CCL19, CCL21, CSAp, HER-2/neu, BrE3, CD1,CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20(e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD55,CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133,CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4, alpha-fetoprotein (AFP),VEGF (e.g., AVASTIN®, fibronectin splice variant), ED-B fibronectin(e.g., L19), EGP-1 (Trop-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1)(e.g., ERBITUX®), ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor,Ga 733, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24,HER-2/neu, histone H2B, histone H3, histone H4, insulin-like growthfactor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R,IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigento which L243 binds, CD66 antigens, i.e., CD66a-d or a combinationthereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophagemigration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac,placental growth factor (PlGF), PSA (prostate-specific antigen), PSMA,PAM4 antigen, PD-1 receptor, PD-L1, NCA-95, NCA-90, A3, A33, Ep-CAM,KS-1, Le(y), mesothelin, 5100, tenascin, TAC, Tn antigen,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, TNF-α, TRAIL receptor (R1 and R2), Trop-2, VEGFR,RANTES, T101, as well as cancer stem cell antigens, complement factorsC3, C3a, C3b, C5a, C5, and an oncogene product.

Cancer stem cells, which are ascribed to be more therapy-resistantprecursor malignant cell populations (Hill and Perris, J. Natl. CancerInst. 2007; 99:1435-40), have antigens that can be targeted in certaincancer types, such as CD133 in prostate cancer (Maitland et al., ErnstSchering Found. Sympos. Proc. 2006; 5:155-79), non-small-cell lungcancer (Donnenberg et al., J. Control Release 2007; 122(3):385-91), andglioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5), and CD44 incolorectal cancer (Dalerba er al., Proc. Natl. Acad. Sci. USA 2007;104(24)10158-63), pancreatic cancer (Li et al., Cancer Res. 2007;67(3):1030-7), and in head and neck squamous cell carcinoma (Prince etal., Proc. Natl. Acad. Sci. USA 2007; 104(3)973-8). Another usefultarget for breast cancer therapy is the LIV-1 antigen described byTaylor et al. (Biochem. J. 2003; 375:51-9).

Checkpoint-inhibitor antibodies have been used in cancer therapy. Immunecheckpoints refer to inhibitory pathways in the immune system that areresponsible for maintaining self-tolerance and modulating the degree ofimmune system response to minimize peripheral tissue damage. However,tumor cells can also activate immune system checkpoints to decrease theeffectiveness of immune response against tumor tissues. Exemplarycheckpoint inhibitor antibodies against cytotoxic T-lymphocyte antigen 4(CTLA4, also known as CD152), programmed cell death protein 1 (PD1, alsoknown as CD279), programmed cell death 1 ligand 1 (PD-L1, also known asCD274) and programmed cell death 1 ligand 2 (PD-L2) (Latchman et al.,2001, Nat Immunol 2:261-8), may be used in combination with one or moreother agents to enhance the effectiveness of immune response againstdisease cells, tissues or pathogens. Exemplary anti-PD1 antibodiesinclude lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558,BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011,CURETECH LTD.). Anti-PD1 antibodies are commercially available, forexample from ABCAM® (AB137132), BIOLEGEND® (EH12.2H7, RMP1-14) andAFFYMETRIX EBIOSCIENCE (J105, J116, MIH4). Exemplary anti-PD-L1antibodies include MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A(GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB). Anti-PD-L1 antibodiesare also commercially available, for example from AFFYMETRIX EBIOSCIENCE(MIH1). Exemplary anti-CTLA4 antibodies include ipilimumab(Bristol-Myers Squibb) and tremelimumab (PFIZER). Anti-PD1 antibodiesare commercially available, for example from ABCAM® (AB134090), SINOBIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE(PA5-29572, PA5-23967, PA5-26465, MA1-12205, MA1-35914). Ipilimumab hasrecently received FDA approval for treatment of metastatic melanoma(Wada et al., 2013, J Transl Med 11:89).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, and colon (e.g., Meyer-Siegler et al.,2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma52:1446-54). Milatuzumab (hLL1) is an exemplary anti-CD74 antibody oftherapeutic use for treatment of MIF-mediated diseases.

Various other antibodies of use are known in the art (e.g., U.S. Pat.Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ. No.20060193865; each incorporated herein by reference.)

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including tumor-associated antigens, have been deposited at theATCC and/or have published variable region sequences and are availablefor use in the claimed methods and compositions. See, e.g., U.S. Pat.Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040; 6,451,310;6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953; 5,525,338. These are exemplary only anda wide variety of other antibodies and their hybridomas are known in theart. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Stickler et al.,2011). It has been reported that G1m1 antibodies contain allotypicsequences that tend to induce an immune response when administered tonon-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al.,2011). Non-G1m1 allotype antibodies are not as immunogenic whenadministered to G1m1 patients (Stickler et al., 2011).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown belowfor the exemplary antibodies rituximab (SEQ ID NO:7) and veltuzumab (SEQID NO:8).

Rituximab heavy chain variable region sequence (SEQ ID NO: 7)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab homy chain variable region(SEQ ID NO: 8) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete allotype 214 (allotype) 356/358 (allotype)431 (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R 3 E/M— A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Nanobodies

Nanobodies are single-domain antibodies of about 12-15 kDa in size(about 110 amino acids in length). Nanobodies can selectively bind totarget antigens, like full-size antibodies, and have similar affinitiesfor antigens. However, because of their much smaller size, they may becapable of better penetration into solid tumors. The smaller size alsocontributes to the stability of the nanobody, which is more resistant topH and temperature extremes than full size antibodies (Van Der Linden etal., 1999, Biochim Biophys Act 1431:37-46). Single-domain antibodieswere originally developed following the discovery that camelids (camels,alpacas, llamas) possess fully functional antibodies without lightchains (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77:13-22).The heavy-chain antibodies consist of a single variable domain (V_(HH))and two constant domains (C_(H)2 and C_(H)3). Like antibodies,nanobodies may be developed and used as multivalent and/or bispecificconstructs. Humanized forms of nanobodies are in commercial developmentthat are targeted to a variety of target antigens, such as IL-6R, vWF,TNF, RSV, RANKL, IL-17A & F and IgE (e.g., ABLYNX®, Ghent, Belgium),with potential clinical use in cancer and other disorders (e.g., Saerenset al., 2008, Curr Opin Pharmacol 8:600-8; Muyldermans, 2013, Ann RevBiochem 82:775-97; Ibanez et al., 2011, J Infect Dis 203:1063-72).

The plasma half-life of nanobodies is shorter than that of full-sizeantibodies, with elimination primarily by the renal route. Because theylack an Fc region, they do not exhibit complement dependentcytotoxicity.

Nanobodies may be produced by immunization of camels, llamas, alpacas orsharks with target antigen, following by isolation of mRNA, cloning intolibraries and screening for antigen binding. Nanobody sequences may behumanized by standard techniques (e.g., Jones et al., 1986, Nature 321:522, Riechmann et al., 1988, Nature 332: 323, Verhoeyen et al., 1988,Science 239: 1534, Carter et al., 1992, Proc. Nat'l Acad. Sci. USA 89:4285, Sandhu, 1992, Crit. Rev. Biotech. 12: 437, Singer et al., 1993, J.Immun. 150: 2844). Humanization is relatively straight-forward becauseof the high homology between camelid and human FR sequences.

In various embodiments, the subject antibodies may comprise nanobodiesfor targeted delivery of conjugated diagnostic agent(s) to targetedcancer cells. Nanobodies of use are disclosed, for example, in U.S. Pat.Nos. 7,807,162; 7,939,277; 8,188,223; 8,217,140; 8,372,398; 8,557,965;8,623,361 and 8,629,244, the Examples section of each incorporatedherein by reference.)

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab′)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. Nos.4,704,692, 4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEBVol 9:73-80 (1995) and R. E. Bird and B. W. Walker, Single ChainAntibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional VH-VL pairs. (Muyldermanset al., 2001). Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may beimmunized with known antigens, such as TNF-α, and VHHs can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca VHH coding sequences have beenidentified and may be used to construct alpaca VHH phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 kDfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

General Techniques for Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding V_(κ) (variable light chain) and V_(H) (variableheavy chain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of a MAb from a cell that expressesa murine MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the MAb may be amplified using the primersVK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primer setdescribed by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human MAb. Alternatively, the Vκ and V_(H)expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Bispecific and Multispecific Antibodies

In certain alternative embodiments, the anti-Trop-2 antibody or fragmentthereof may be co-administered with, for example, a hapten-bindingantibody or fragment thereof, such as an anti-HSG or anti-In-DTPAantibody. Such bispecific antibodies may be of use in pretargetingtechniques for administration of diagnostic and/or therapeutic agents toTrop-2 positive tumors in vivo. In other embodiments, bispecific ormultispecific antibodies may be utilized directly for anti-cancertherapy.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Other methods include improving theefficiency of generating hybrid hybridomas by gene transfer of distinctselectable markers via retrovirus-derived shuttle vectors intorespective parental hybridomas, which are fused subsequently (DeMonte,et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of ahybridoma cell line with expression plasmids containing the heavy andlight chain genes of a different antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv), as discussedabove. Reduction of the peptide linker length to less than 12 amino acidresidues prevents pairing of V_(H) and V_(L) domains on the same chainand forces pairing of V_(H) and V_(L) domains with complementary domainson other chains, resulting in the formation of functional multimers.Polypeptide chains of V_(H) and V_(L) domains that are joined withlinkers between 3 and 12 amino acid residues form predominantly dimers(termed diabodies). With linkers between 0 and 2 amino acid residues,trimers (termed triabody) and tetramers (termed tetrabody) are favored,but the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of V-domains (V_(H)-linker-V_(L)or V_(L)-linker-V_(H)), in addition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “DOCKANDLOCK®” (DNL®), discussed inmore detail below, has been utilized to produce combinations ofvirtually any desired antibodies, antibody fragments and other effectormolecules. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

Dock-and-Lock® (DNL®)

Bispecific or multispecific antibodies or other constructs may beproduced using the DOCK-AND-LOCK® technology (see, e.g., U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400, the Examplessection of each incorporated herein by reference). Generally, thetechnique takes advantage of the specific and high-affinity bindinginteractions that occur between a dimerization and docking domain (DDD)sequence of the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and an anchor domain (AD) sequence derived from any of a varietyof AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wongand Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and ADpeptides may be attached to any protein, peptide or other molecule,preferably as a fusion protein comprising the AD or DDD sequence.Because the DDD sequences spontaneously dimerize and bind to the ADsequence, the technique allows the formation of complexes between anyselected molecules that may be attached to DDD or AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα and RIIβThe R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL®complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL®constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described below, virtually any proteinor peptide may be incorporated into a DNL® construct. However, thetechnique is not limiting and other methods of conjugation may beutilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNIL® constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 9) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 10) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 11) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 12)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 13) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 14) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 15) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL® complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 16)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEE AK PKA RIβ(SEQ ID NO: 17) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 18) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 19) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:9 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 9) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:9are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:20 to SEQ ID NO:39 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 2Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 9). Consensussequence disclosed as SEQ ID NO: 94. S H I Q I P P G L T E L L Q G Y T VE V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F T R L R E AR A N N E D L D S K K D L K L I I I V V VTHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 31)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 32)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 33)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 34)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 35)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 36)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 37)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 38)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 39)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:11), witha binding constant for DDD of 0.4 nM. The AKAP-IS sequence was designedas a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:11 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:19), similar tothat shown for DDD1 (SEQ ID NO:16) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:40 to SEQ ID NO:57 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 1) QIEYLAKQIVDNAIQQA

TABLE 3 Conservative Amino Acid Substitutions in AD1 (SEQ IDNO: 11). Consensus sequence disclosed as SEQ ID NO: 95. Q I E Y L A K QI V D N A I Q Q A N L D F I R N E Q N N L V T V I S VNIEYLAKQIVDNAIQQA (SEQ ID NO: 40) QLEYLAKQIVDNAIQQA (SEQ ID NO: 41)QVEYLAKQIVDNAIQQA (SEQ ID NO: 42) QIDYLAKQIVDNAIQQA (SEQ ID NO: 43)QIEFLAKQIVDNAIQQA (SEQ ID NO: 44) QIETLAKQIVDNAIQQA (SEQ ID NO: 45)QIESLAKQIVDNAIQQA (SEQ ID NO: 46) QIEYIAKQIVDNAIQQA (SEQ ID NO: 47)QIEYVAKQIVDNAIQQA (SEQ ID NO: 48) QIEYLARQIVDNAIQQA (SEQ ID NO: 49)QIEYLAKNIVDNAIQQA (SEQ ID NO: 50) QIEYLAKQIVENAIQQA (SEQ ID NO: 51)QIEYLAKQIVDQAIQQA (SEQ ID NO: 52) QIEYLAKQIVDNAINQA (SEQ ID NO: 53)QIEYLAKQIVDNAIQNA (SEQ ID NO: 54) QIEYLAKQIVDNAIQQL (SEQ ID NO: 55)QIEYLAKQIVDNAIQQI (SEQ ID NO: 56) QIEYLAKQIVDNAIQQV (SEQ ID NO: 57)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:58),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL® constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:59-61.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:12, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 58) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 59) QIEYKAKQIVDHAIHQA(SEQ ID NO: 60) QIEYHAKQIVDHAIHQA (SEQ ID NO: 61) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

Ru-Specific AKAPs AKAP-KL (SEQ ID NO: 62) PLEYQAGLLVQNAIQQAI AKAP 79(SEQ ID NO: 63) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 64)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 65)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 66) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 67) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 68) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 69)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 70) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 71) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:72-74. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:72), RIAD (SEQ IDNO:73) and PV-38 (SEQ ID NO:74). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 72) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 73)LEQYANQLADQIIKEATE PV-38

FEELAWKIAKMIWSDVFQQC (SEQ ID NO:74)

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 11) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 75) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 76) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 77) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 78) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 79) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 80) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 81) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 82) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 83) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 84) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 85) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 86) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 87) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 88) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 89) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 90) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 91) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 92)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:11). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO:11) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:9. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 9) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:9) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 9). Consensussequence disclosed as SEQ ID NO: 96. S H I Q I P P G L T E L L Q G Y T VE V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A R A N L D SK K L L I I I V V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Alternative DNL® Structures

In certain alternative embodiments, DNL® constructs may be formed usingalternatively constructed antibodies or antibody fragments, in which anAD moiety may be attached at the C-terminal end of the kappa light chain(C_(k)), instead of the C-terminal end of the Fc on the heavy chain. Thealternatively formed DNL® constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. Nos. 61/654,310, filed Jun. 1,2012, 61/662,086, filed Jun. 20, 2012, 61/673,553, filed Jul. 19, 2012,and 61/682,531, filed Aug. 13, 2012, the entire text of eachincorporated herein by reference. The light chain conjugated DNL®constructs exhibit enhanced Fc-effector function activity in vitro andimproved pharmacokinetics, stability and anti-lymphoma activity in vivo(Rossi et al., 2013, Bioconjug Chem 24:63-71).

C_(k)-conjugated DNL® constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. Nos. 61/654,310, 61/662,086,61/673,553, and 61/682,531. Briefly, C_(k)-AD2-IgG, was generated byrecombinant engineering, whereby the AD2 peptide was fused to theC-terminal end of the kappa light chain. Because the natural C-terminusof C_(K) is a cysteine residue, which forms a disulfide bridge toC_(H)1, a 16-amino acid residue “hinge” linker was used to space the AD2from the C_(K)-V_(H)1 disulfide bridge. The mammalian expression vectorsfor C_(k)-AD2-IgG-veltuzumab and C_(k)-AD2-IgG-epratuzumab wereconstructed using the pdHL2 vector, which was used previously forexpression of the homologous C_(H)3-AD2-IgG modules. A 2208-bpnucleotide sequence was synthesized comprising the pdHL2 vector sequenceranging from the Bam HI restriction site within the V_(K)/C_(K) intronto the Xho I restriction site 3′ of the C_(k) intron, with the insertionof the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ IDNO:93) and AD2, in frame at the 3′end of the coding sequence for C_(K).This synthetic sequence was inserted into the IgG-pdHL2 expressionvectors for veltuzumab and epratuzumab via Bam HI and Xho I restrictionsites. Generation of production clones with SpESFX-10 were performed asdescribed for the C_(H)3-AD2-IgG modules. C_(k)-AD2-IgG-veltuzumab andC_(k)-AD2-IgG-epratuzumab were produced by stably-transfected productionclones in batch roller bottle culture, and purified from the supernatantfluid in a single step using Mab Select (GE Healthcare) Protein Aaffinity chromatography.

Following the same DNIL® process described previously for 22-(20)-(20)(Rossi et al., 2009, Blood 113:6161-71), C_(k)-AD2-IgG-epratuzumab wasconjugated with C_(H)1-DDD2-Fab-veltuzumab, a Fab-based module derivedfrom veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the 22*indicates the C_(k)-AD2 module of epratuzumab and each (20) symbolizes astabilized dimer of veltuzumab Fab. The properties of 22*-(20)-(20) werecompared with those of 22-(20)-(20), the homologous Fc-bsHexAbcomprising C_(H)3-AD2-IgG-epratuzumab, which has similar composition andmolecular size, but a different architecture.

Following the same DNL® process described previously for 20-2b (Rossi etal., 2009, Blood 114:3864-71), C_(k)-AD2-IgG-veltuzumab, was conjugatedwith IFNα2b-DDD2, a module of IFNα2b with a DDD2 peptide fused at itsC-terminal end, to generate 20*-2b, which comprises veltuzumab with adimeric IFNα2b fused to each light chain. The properties of 20*-2b werecompared with those of 20-2b, which is the homologous Fc-IgG-IFNα.

Each of the bsHexAbs and IgG-IFNα were isolated from the DNIL® reactionmixture by MabSelect affinity chromatography. The two C_(k)-derivedprototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprisingepratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and aCD20-targeting immunocytokine, comprising veltuzumab and four moleculesof interferon-α2b, displayed enhanced Fc-effector functions in vitro, aswell as improved pharmacokinetics, stability and anti-lymphoma activityin vivo, compared to their Fc-derived counterparts.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL® constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Pre-Targeting

Bispecific or multispecific antibodies may be of use in pretargetingtechniques. In this case, one or more diagnostic and/or therapeuticagents may be conjugated to a targetable construct that comprises one ormore haptens. The hapten is recognized by at least one arm of abispecific or multispecific antibody that also binds to atumor-associated antigen or other disease-associated antigen. In thiscase, the therapeutic agent binds indirectly to the antibodies, via thebinding of the targetable construct. This process is referred to aspretargeting.

Pre-targeting is a multistep process originally developed to resolve theslow blood clearance of directly targeting antibodies, which contributesto undesirable toxicity to normal tissues such as bone marrow. Withpre-targeting, a therapeutic agent is attached to a small deliverymolecule (targetable construct) that is cleared within minutes from theblood. A pre-targeting bispecific or multispecific antibody, which hasbinding sites for the targetable construct as well as a target antigen,is administered first, free antibody is allowed to clear fromcirculation and then the targetable construct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of diagnosing or treating a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents.

Targetable Constructs

In certain embodiments, targetable construct peptides labeled with oneor more therapeutic or diagnostic agents for use in pre-targeting may beselected to bind to a bispecific antibody with one or more binding sitesfor a targetable construct peptide and one or more binding sites for atarget antigen associated with a disease or condition. Bispecificantibodies may be used in a pretargeting technique wherein the antibodymay be administered first to a subject. Sufficient time may be allowedfor the bispecific antibody to bind to a target antigen and for unboundantibody to clear from circulation. Then a targetable construct, such asa labeled peptide, may be administered to the subject and allowed tobind to the bispecific antibody and localize at the diseased cell ortissue.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons. More usually, the targetable construct peptide will have fouror more residues and one or more haptens for binding, e.g., to abispecific antibody. Exemplary haptens may include In-DTPA(indium-diethylene triamine pentaacetic acid) or HSG (histamine succinylglycine). The targetable construct may also comprise one or morechelating moieties, such as DOTA(1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid), NOTA(1,4,7-triaza-cyclononane-1,4,7-triacetic acid), TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), NETA([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]aceticacid) or other known chelating moieties. Chelating moieties may be used,for example, to bind to a therapeutic and or diagnostic radionuclide,paramagnetic ion or contrast agent.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids orpeptoids may be used.

The peptides used as targetable constructs are conveniently synthesizedon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later forconjugation of chelating moieties or other agents, are advantageouslyblocked with standard protecting groups such as a Boc group, whileN-terminal residues may be acetylated to increase serum stability. Suchprotecting groups are well known to the skilled artisan. See Greene andWuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons,N.Y.). When the peptides are prepared for later use within thebispecific antibody system, they are advantageously cleaved from theresins to generate the corresponding C-terminal amides, in order toinhibit in vivo carboxypeptidase activity.

Where pretargeting with bispecific antibodies is used, the antibody willcontain a first binding site for an antigen produced by or associatedwith a target tissue and a second binding site for a hapten on thetargetable construct. Exemplary haptens include, but are not limited to,HSG and In-DTPA. Antibodies raised to the HSG hapten are known (e.g. 679antibody) and can be easily incorporated into the appropriate bispecificantibody (see, e.g., U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644,incorporated herein by reference with respect to the Examples sections).However, other haptens and antibodies that bind to them are known in theart and may be used, such as In-DTPA and the 734 antibody (e.g., U.S.Pat. No. 7,534,431, the Examples section incorporated herein byreference).

Immunoconjugates

Various embodiments may involve use of immunoconjugates, comprising ananti-Trop-2 antibody or antigen-binding fragment thereof attached to oneor more diagnostic or therapeutic agents. In some embodiments, a drug orother agent may be attached to an antibody or fragment thereof via acarrier moiety. Carrier moieties may be attached, for example to reducedSH groups and/or to carbohydrate side chains. A carrier moiety can beattached at the hinge region of a reduced antibody component viadisulfide bond formation. Alternatively, such agents can be attachedusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the carrier moiety canbe conjugated via a carbohydrate moiety in the Fc region of theantibody.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component is an antibodyfragment. However, it is possible to introduce a carbohydrate moietyinto the light chain variable region of a full length antibody orantibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919(1995); U.S. Pat. Nos. 5,443,953 and 6,254,868, the Examples section ofwhich is incorporated herein by reference. The engineered carbohydratemoiety is used to attach the therapeutic or diagnostic agent.

An alternative method for attaching carrier moieties to a targetingmolecule involves use of click chemistry reactions. The click chemistryapproach was originally conceived as a method to rapidly generatecomplex substances by joining small subunits together in a modularfashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31;Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistryreaction are known in the art, such as the Huisgen 1,3-dipolarcycloaddition copper catalyzed reaction (Tornoe et al., 2002, J OrganicChem 67:3057-64), which is often referred to as the “click reaction.”Other alternatives include cycloaddition reactions such as theDiels-Alder, nucleophilic substitution reactions (especially to smallstrained rings like epoxy and aziridine compounds), carbonyl chemistryformation of urea compounds and reactions involving carbon-carbon doublebonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach carrier moieties to antibodiesin vitro.

Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that arecombinant glycoprotein expressed in CHO cells in the presence ofperacetylated N-azidoacetylmannosamine resulted in the bioincorporationof the corresponding N-azidoacetyl sialic acid in the carbohydrates ofthe glycoprotein. The azido-derivatized glycoprotein reactedspecifically with a biotinylated cyclooctyne to form a biotinylatedglycoprotein, while control glycoprotein without the azido moietyremained unlabeled (Id.) Laughlin et al. (2008, Science 320:664-667)used a similar technique to metabolically label cell-surface glycans inzebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localization in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Antibody labeling techniques using biological incorporation of labelingmoieties are further disclosed in U.S. Pat. No. 6,953,675 (the Examplessection of which is incorporated herein by reference). Such “landscaped”antibodies were prepared to have reactive ketone groups on glycosylatedsites. The method involved expressing cells transfected with anexpression vector encoding an antibody with one or more N-glycosylationsites in the CH1 or Vκ domain in culture medium comprising a ketonederivative of a saccharide or saccharide precursor. Ketone-derivatizedsaccharides or precursors included N-levulinoyl mannosamine andN-levulinoyl fucose. The landscaped antibodies were subsequently reactedwith agents comprising a ketone-reactive moiety, such as hydrazide,hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeledtargeting molecule. Exemplary agents attached to the landscapedantibodies included chelating agents like DTPA, large drug moleculessuch as doxorubicin-dextran, and acyl-hydrazide containing peptides. Thelandscaping technique is not limited to producing antibodies comprisingketone moieties, but may be used instead to introduce a click chemistryreactive group, such as a nitrone, an azide or a cyclooctyne, onto anantibody or other biological molecule.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above.

Alternatively, methods of chemical conjugation of such moieties tobiomolecules are well known in the art, and any such known method may beutilized. General methods of immunoconjugate formation are disclosed,for example, in U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338;5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393;6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and 7,259,240,the Examples section of each incorporated herein by reference.

Diagnostic Agents

Diagnostic agents may comprise any detectable agent that may be used tolabel a detection antibody, or to directly label a CTC, and arepreferably selected from the group consisting of a radionuclide, aradiological contrast agent, a paramagnetic ion, a metal, a fluorescentlabel, a chemiluminescent label, an ultrasound contrast agent and aphotoactive agent. Such diagnostic agents are well known and any suchknown diagnostic agent may be used. Non-limiting examples of diagnosticagents may include a radionuclide such as ¹¹¹In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F,⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O,¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸⁷Sr, orother gamma-, beta-, or positron-emitters. Paramagnetic ions of use mayinclude chromium (III), manganese (II), iron (III), iron (II), cobalt(II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) or erbium (III). Metal contrast agentsmay include lanthanum (III), gold (III), lead (II) or bismuth (III).Ultrasound contrast agents may comprise liposomes, such as gas filledliposomes. Radiopaque diagnostic agents may be selected from compounds,barium compounds, gallium compounds, and thallium compounds.

In certain embodiments, the fluorescent probe may be a DYLIGHT® dye(Thermo Fisher Scientific, Rockford, Ill.). The DYLIGHT® dye series arehighly polar (hydrophilic), compatible with aqueous buffers, photostableand exhibit high fluorescence intensity. They remain highly fluorescentover a wide pH range and are preferred for various applications.However, the skilled artisan will realize that a variety of fluorescentdyes are known and/or are commercially available and may be utilized.Other fluorescent agents include, but are not limited to, dansylchloride, rhodamine isothiocyanate, Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole),Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet,cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid,erythrosine, phthalocyanines, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, and Texas Red. (See, e.g., U.S. Pat. Nos.5,800,992; 6,319,668.) These and other luminescent labels may beobtained from commercial sources such as Molecular Probes (Eugene,Oreg.), and EMD Biosciences (San Diego, Calif.).

Chemiluminescent labels of use may include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt or anoxalate ester.

Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially with an anti-Trop-2 or other anti-TAA antibody.Alternatively, such agents may be conjugated to antibodies, for example,drugs, toxins, oligonucleotides, immunomodulators, hormones, hormoneantagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesisinhibitors, etc. Therapeutic agents include, for example, cytotoxicdrugs such as vinca alkaloids, anthracyclines such as doxorubicin,2-PDox or pro-2-PDox, gemcitabine, epipodophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, SN-38, COX-2inhibitors, antimitotics, anti-angiogenic and pro-apoptotic agents,particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinaseinhibitors, and others. Other useful anti-cancer cytotoxic drugs includenitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, COX-2 inhibitors, antimetabolites, pyrimidine analogs, purineanalogs, platinum coordination complexes, mTOR inhibitors, tyrosinekinase inhibitors, proteosome inhibitors, HDAC inhibitors,camptothecins, hormones, and the like. Suitable cytotoxic agents aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitable cytotoxicagents, such as experimental drugs, are known to those of skill in theart. In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to anti-Trop-2 or otheranti-TAA antibodies. In another preferred embodiment, gemcitabine isadministered to the subject in conjunction with SN-38-hRS7 and/or⁹⁰Y-hPAM4.

A toxin can be of animal, plant or microbial origin. Toxins of useinclude ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, onconase, gelonin, diphtheriatoxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, forexample, Pastan et al., Cell 47:641 (1986), Goldenberg, C A—A CancerJournal for Clinicians 44:43 (1994), Sharkey and Goldenberg, CA—A CancerJournal for Clinicians 56:226 (2006). Additional toxins suitable for useare known to those of skill in the art and are disclosed in U.S. Pat.No. 6,077,499, the Examples section of which is incorporated herein byreference.

As used herein, the term “immunomodulator” includes a cytokine, alymphokine, a monokine, a stem cell growth factor, a lymphotoxin, ahematopoietic factor, a colony stimulating factor (CSF), an interferon(IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, a transforming growth factor (TGF), TGF-α, TGF-β,insulin-like growth factor (ILGF), erythropoietin, thrombopoietin, tumornecrosis factor (TNF), TNF-α, TNF-β, a mullerian-inhibiting substance,mouse gonadotropin-associated peptide, inhibin, activin, vascularendothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, interferon-λ, S1 factor, IL-1, IL-1cc, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18 IL-21 and IL-25, LIF, kit-ligand,FLT-3, angiostatin, thrombospondin, endostatin, lymphotoxin, and thelike.

Particularly useful therapeutic radionuclides include, but are notlimited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²²⁷Th, and²¹¹Pb. The therapeutic radionuclide preferably has a decay energy in therange of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for anAuger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV foran alpha emitter. Maximum decay energies of usefulbeta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Fm-255 and Th-227. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

For example, ⁹⁰Y, which emits an energetic beta particle, can be coupledto an antibody, antibody fragment or fusion protein, usingdiethylenetriaminepentaacetic acid (DTPA), or more preferably usingDOTA. Methods of conjugating ⁹⁰Y to antibodies or targetable constructsare known in the art and any such known methods may be used. (See, e.g.,U.S. Pat. No. 7,259,249, the Examples section of which is incorporatedherein by reference. See also Linden et al., Clin Cancer Res.11:5215-22, 2005; Sharkey et al., J Nucl Med. 46:620-33, 2005; Sharkeyet al., J Nucl Med. 44:2000-18, 2003.)

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In ⁹⁵Ru 97Ru ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ¹²⁵Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³P, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe,⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated antibody or antibody fragment. For example, theradiosensitizer can be used in combination with a radiolabeled antibodyor antibody fragment. The addition of the radiosensitizer can result inenhanced efficacy when compared to treatment with the radiolabeledantibody or antibody fragment alone. Radiosensitizers are described inD. M. Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRCPress (1995). Other typical radionsensitizers of interest for use withthis technology include gemcitabine, 5-fluorouracil, and cisplatin, andhave been used in combination with external irradiation in the therapyof diverse cancers.

Antibodies or fragments thereof that have a boron addend-loaded carrierfor thermal neutron activation therapy will normally be affected insimilar ways. However, it will be advantageous to wait untilnon-targeted immunoconjugate clears before neutron irradiation isperformed. Clearance can be accelerated using an anti-idiotypic antibodythat binds to the anti-cancer antibody. See U.S. Pat. No. 4,624,846 fora description of this general principle. For example, boron addends suchas carboranes, can be attached to antibodies. Carboranes can be preparedwith carboxyl functions on pendant side chains, as is well-known in theart. Attachment of carboranes to a carrier, such as aminodextran, can beachieved by activation of the carboxyl groups of the carboranes andcondensation with amines on the carrier. The intermediate conjugate isthen conjugated to the antibody. After administration of the antibodyconjugate, a boron addend is activated by thermal neutron irradiationand converted to radioactive atoms which decay by alpha-emission toproduce highly toxic, short-range effects.

Formulation and Administration

Where therapeutic antibodies are to be administered in vivo, suitableroutes of administration may include, without limitation, oral,parenteral, rectal, transmucosal, intestinal administration,intramedullary, intrathecal, direct intraventricular, intravenous,intravitreal, intracavitary, intraperitoneal, or intratumoralinjections. The preferred routes of administration are parenteral, morepreferably intravenous. Alternatively, one may administer the compoundin a local rather than systemic manner, for example, via injection ofthe compound directly into a solid or hematological tumor.

Antibodies can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the antibody is combinedin a mixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

In a preferred embodiment, the antibody is formulated in Good'sbiological buffer (pH 6-7), using a buffer selected from the groupconsisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The antibody can be formulated for intravenous administration via, forexample, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the antibody. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio Technology 10: 1446 (1992). The rate of release ofan antibody from such a matrix depends upon the molecular weight of theantibody, the amount of antibody within the matrix, and the size ofdispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

Generally, the dosage of an administered antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of antibody that is inthe range of from about 0.3 mg/kg to 5 mg/kg as a single intravenousinfusion, although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 0.3-5 mg/kg for a 70 kg patient, forexample, is 21-350 mg, or 12-206 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 2-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy. Preferred dosages may include, but are not limited to, 0.3mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 2.0 mg/kg,2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, and 5.0 mg/kg.More preferred dosages are 0.6 mg/kg for weekly administration and 1.2mg/kg for less frequent dosing. Any amount in the range of 0.3 to 5mg/kg may be used. The dosage is preferably administered multiple times,once a week. A minimum dosage schedule of 4 weeks, more preferably 8weeks, more preferably 16 weeks or longer may be used, with the dosefrequency dependent on toxic side-effects and recovery therefrom, mostlyrelated to hematological toxicities. The schedule of administration maycomprise administration once or twice a week, on a cycle selected fromthe group consisting of: (i) weekly; (ii) every other week; (iii) oneweek of therapy followed by two, three or four weeks off; (iv) two weeksof therapy followed by one, two, three or four weeks off; (v) threeweeks of therapy followed by one, two, three, four or five week off;(vi) four weeks of therapy followed by one, two, three, four or fiveweek off; (vii) five weeks of therapy followed by one, two, three, fouror five week off; and (viii) monthly. The cycle may be repeated 2, 4, 6,8, 10, or 12 times or more.

Alternatively, an antibody may be administered as one dosage every 2 or3 weeks, repeated for a total of at least 3 dosages. Or, twice per weekfor 4-6 weeks. The dosage may be administered once every other week oreven less frequently, so the patient can recover from any drug-relatedtoxicities. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. The dosing schedule can optionally berepeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. In preferredembodiments, the method of the invention is used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

Kits

Various embodiments may concern kits containing components suitable fordetecting Trop-2 positive CTCs in a patient. Exemplary kits may containat least one anti-Trop-2 antibody as described herein. In certainembodiments, the antibody may be conjugated to at least one diagnosticagent. In alternative embodiments, a second antibody that binds to aTrop-2 positive CTC may be included. The second antibody may bind to adifferent epitope of Trop-2 or to a different TAA, and may be labeledwith at least one diagnostic agent. In certain embodiments, ananti-Trop-2 antibody or antigen binding fragment thereof may be providedin the form of a prefilled syringe or vial containing a sterile, liquidformulation or lyophilized preparation of antibody (e.g., Kivitz et al.,Clin. Ther. 2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions for use of the kit.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims.

Example 1. Cell Binding Assay of Anti-Trop-2 Antibodies

Two different murine monoclonal antibodies against human Trop-2 wereobtained. The first, 162-46.2, was purified from a hybridoma (ATCC,HB-187) grown up in roller-bottles. A second antibody, MAB650, waspurchased from R&D Systems (Minneapolis, Minn.). For a comparison ofbinding, the Trop-2-positive human gastric carcinoma, NCI-N87, was usedas the target. Cells (1.5×10⁵/well) were plated into 96-well plates theday before the binding assay. The following morning, a dose/responsecurve was generated with 162-46.2, MAB650, and murine RS7 (0.03 to 66nM) (not shown). These primary antibodies were incubated with the cellsfor 1.5 h at 4° C. Wells were washed and an anti-mouse-HRP secondaryantibody was added to all the wells for 1 h at 4° C. Wells are washedagain followed by the addition of a luminescence substrate. Plates wereread using Envision plate reader and values are reported as relativeluminescent units.

All three antibodies had similar K_(D)-values of 0.57 nM for RS7, 0.52nM for 162-46.2 and 0.49 nM for MAB650 (not shown). However, whencomparing the maximum binding (B_(max)) of 162-46.2 and MAB650 to RS7they were reduced by 25% and 50%, respectively (B_(Max) 11,250 for RS7,8,471 for 162-46.2 and 6,018 for MAB650) indicating different bindingproperties in comparison to RS7 (not shown).

Example 2. Collection and Storage of Blood Samples

Ten mL blood samples are drawn from each of 10 healthy donors and 20patients with metastatic breast cancer and dispensed into a CELLSAVE™Preservative tube (Jassen Diagnostics LLC, Raritan, N.J.). The samplesare stored at RT and processed within 72 h of blood collection (Allardet al., 2004, Clin Cancer Res, 10: 6897). Alternatively, 10 mL of bloodsamples are drawn into a CYTOCHEX® Blood collection tube (Streck, Omaha,Nebr.), maintained at RT, and processed within 7 days (Ng et al., 2012 JImmunol Methods, 385: 79). Blood can also be drawn into 10 mL K2EDTAVACUTAINER® (BD, Waltham, Mass.), fixed with the LIQUIDBIOPSY® fixative(Cynvenio Biosystems, Westlake Village, Calif.)) within 4 h ofcollection, stored at room temperature, and processed within 96 h offixation.

Example 3. Spiking of Cancer Cells in Blood Samples from Healthy Donors

SK-BR-3 and BxPC-3 cells, both expressing high levels of Trop-2, arecultured in their designated medium, and harvested using trypsin. Theviability and cell number of the resulting cell suspensions are assessedby Guava EASYCYTEυ flow cytometer. The cell suspensions are used onlywhen their viability exceeds 90%. The number of cells spiked into normalserum is from 1 to 100 per mL. Cancer cells that express moderate levelsof Trop-2, for example, MCF-7, LoVo, and LS 174 T, low levels of Trop-2,for example, HT-29, or are negative for Trop-2, for example, A549 andH460, can also be used to spike blood samples.

Example 4. Isolation of Epithelial Cancer Cells from Spiked BloodSamples with the Use of a Magnetic Device

Blood samples spiked with epithelial cancer cells are incubated withbiotinylated tri-Fab hRS7 (biotin-E1/3, prepared by the DNL® techniquedescribed above), and ferrofluids coated with streptavidin (FF-SV) toimmunomagnetically enrich epithelial cells. Briefly, 7.5 mL of a bloodsample containing a known number of spiked BxPC-3 or SK-BR-3 are mixedwith 6 mL of buffer, and centrifuged at 800×g for 10 min. After removingthe plasma and buffer layer, biotin-E1/3 and FF-SV are added andincubated for 1 h. Subsequently, unlabeled cells are removed fromlabeled cells following magnetic separation. Cells labeled withbiotin-E1/3 are then detached from FF-SV with a further wash andcentrifugation, and are analyzed by flow cytometry after labeling withDAPI, PE-anti-CK18, and APC-anti-CD45. Nucleated cells lacking CD45 andexpressing cytokeratin (CK8, CK18, CK19) are generally defined as CTCs(Swaby & Cristofanilli, 2011, BMC Medicine, 9: 43).

Example 5. Isolation of Epithelial Cancer Cells from Spiked BloodSamples without the Use of a Magnetic Device

Blood samples spiked with epithelial cancer cells are incubated withbiotin-E1/3 in a microvortex-generating herringbone-chip (HP-Chip)chemically modified with avidin as described by Stott et al (2010, PNAS,107: 18392), or more preferably, are incubated with biotin-E1/3 for 1 hbefore adding to NanoVelcro chips functionalized with streptavidin asdescribed by Lu et al. (2013, Methods, 64: 144). After rinsing away theunbound cells, the bound cells are analyzed for CTCs as described inExample 3.

Example 6. Detection of Epithelial Cancer Cells from Spiked BloodSamples without Prior Enrichment

Red blood cells in blood samples spiked with BxPC-3 are lysed withammonium chloride, and centrifuged. The cell pellets are collected andincubated with FITC-labeled E1/3. The live cells in suspension are thenapplied to a poly-lysine-treated slide and analyzed with a laserscanning cytometer (Pachmann et al., 2005, Breast Cancer Res, 7: R975).Alternatively, the cell pellets collected after lysis of red blood cellsare incubated with a cocktail comprising biotinylated E1/3 and one ormore of other biotinylated DNL® conjugates in the presence ofFITC-labeled avidin. The live cells in suspension are then analyzed bylaser scanning cytometer.

Example 7. Detection of Epithelial Cancer Cells from Spiked BloodSamples Using a Bispecific Construct Targeting Both Trop-2 and EGFR

Blood samples spiked with BxPC-3 cells, which express high levels ofboth Trop-2 and EGFR, are incubated with a biotinylated bispecificTri-Fab, designated (E1)-225, and ferrofluids coated with streptavidin(FF-SV), as described in Example 4. (E1)-225 is generated by conjugatingC_(H)1-DDD2-Fab-hRS7 to C_(H)1-AD2-Fab-c225, thus providing bivalent andmonovalent binding to Trop-2 and EGFR, respectively. When compared withthe enrichment using only monospecific hRS7 or c225 (cetuximab), thebispecific (E1)-225 is able to capture more BxPC-3 spiked into the bloodsamples, with less contamination of CD45-positive white blood cells.

Example 8. Detection of Trop-2⁺ CTCs Using LIQUIDBIOPSY® System

A LIQUIDBIOPSY® instrument (Cat. No. A28188), LIQUIDBIOPSY® BloodCollection Kit (Cat. No. A28171) and LIQUIDBIOPSY® Reagents andConsumables Kits (Cat. Nos. A28186, A28187) are obtained from LifeTechnologies, ThermoFisher (Grand Island, N.Y.). The LIQUIDBIOPSY® kitsincludes a stabilization protocol for whole-blood samples, allowingunrefrigerated shipping of samples (96-hour window), as well as buffers,reagents, vials, elution tubes, and flow cells to process blood samples.

The humanized RS7 (hRS7) monoclonal antibody (sacituzumab) isbiotinylated using the protocols and reagents provided with the Reagentsand Consumables kit. Biotinylated hRS7 (sacituzumab) is used in place ofthe anti-EpCAM biotinylated antibody provided with the Reagents andConsumables Kit. Alternatively, anti-TROP-2 Biotinylated Antibody (Cat.No. BAF650, R&D Systems, Minneapolis, Minn.) is used in place ofanti-EpCAM.

Circulating tumor cells from the blood of patients with solid tumors areisolated using the anti-TROP-2 biotinylated antibody and the instrumentand reagents discussed above, according to the manufacturer'sinstructions. Isolated tumor cells are released from the slide, andconfirmed by flow cytometry after labeling with DAPI, PE-anti-CK18, andAPC-anti-CD45, as described in Example 3. Released cells from a secondblood specimen are cultured, and a colony of live cells is obtained,which are isolated and analyzed by FISH for the copy number of Trop-2and chromosome-1 using specific probes available from Empire Genomics(Buffalo, N.Y.). FIG. 1 and FIG. 2 are representative results obtainedin MCF-7 (Trop-2-positive) and A549 (Trop-2-negative) cells, showing 3and 2 copies of the Trop-2 gene, respectively. In addition, the copynumber of topoisomerase-I (TOP1) and chromosome-20 are also determinedusing specific probes provided by Abnova (Taipei, Taiwan) anddocumented. FIG. 3 and FIG. 4 are representative results obtained inMCF-7 and A549 cells, showing 7 and 3 copies of the TOP1 gene,respectively. The simultaneous detection and quantitation of copynumbers of Trop-2 and TOP1 allow the determination of cancer cells thatalso express TOP1, which would indicate which patient tumors may beparticularly responsive or resistant to a TOP1-inhibitor therapy, suchas with irinotecan. This is particularly useful when using sacituzumabgovitecan (IMMU-132), which targets Trop-2-expressing cancer cells anddelivers SN-38 selectively to such cells. Recovery of tumor cells fromblood samples is compared using anti-Trop-2 hRS7 antibody versus theanti-EpCAM antibody provided with the kit. Surprisingly, recovery ofCTCs is higher with the anti-Trop-2 antibody than the anti-EpCAMantibody.

Example 9. Isolation of Trop-2⁺ CTCs with IMAG™ Magnetic Particles

Purified mouse anti-human Trop-2 antibody is prepared from clone 162-46,purchased from BD Pharmingen (San Jose, Calif.). The anti-Trop-2antibody is biotinylated as described in Example 7. IMAG™ magneticparticles (Streptavidin Particles Plus-DM) and a BD IMAG™ CellSeparation Magnet are purchased from BD Biosciences (San Jose, Calif.).Ten ml plastic whole blood tubes spray-coated with K2EDTA (Cat. No.366643) are also purchased from BD.

For separation and analysis of CTCs, ten mL blood samples are drawn frompatients with lung cancer and stored in K2EDTA tubes. Mononuclear cellsare obtained by density gradient centrifugation using Ficoll-Hypaquesolution. Protocols for positive selection of CTCs from Ficoll-Hypaqueare as disclosed in BD Technical Data Sheet Streptavidin ParticlesPlus-DM Material Number: 557812. After the final wash step on the BDIMAG™ magnet, the released cells are resuspended in buffer.

The cells are stained with fluorescently labeled anti-cytokeratin,fluorescently labeled affinity purified goat anti-TROP-2, DAPI and/oranti-CD45. Subsequent immunofluorescence images are taken of thecaptured cells, followed by comprehensive computer aided analysis basedon fluorescence intensities and cell morphology.

Example 10. Detection of Trop-2⁺ CTCs and Treatment of Metastatic Trop-2Expressing Cancer

A CELLSEARCH® system and Circulating Tumor Cell Kit are obtained VeridexLLC (Raritan, N.J.). A 7.5 ml blood sample is collected from a 65year-old male with suspected NSCLC and stored in a CellSave tube(Veridex LLC). The anti-Trop-2 hRS7 antibody is substituted for theanti-EpCAM antibody provided with the CELLSEARCH® kit. The blood sampleis mixed with magnetic nanoparticles conjugated to anti-Trop-2 antibody.Cells are stained with fluorescently labeled anti-CD45 and anti-CKantibodies and cell nuclei are fluorescently labeled with DAPI nucleardye. A strong magnetic field is generated in the CELLSEARCH® system andused to separate cells bound to the magnetic nanoparticles, which arethen analyzed by FISH to determine Trop-2 copy number, as described inExample 7 above. The results show the presence of circulating Trop-2⁺tumor cells, with 4 copies of Trop-2 per cell. The presence of high copynumbers of Trop-2 in the CTCs indicates that the patient is a goodcandidate for therapy with anti-Trop-2 antibodies.

Further clinical workup shows the presence of stage IIIB NSCLC (squamouscell carcinoma). Initial treatment of caboplatin/etoposide (3 mo) inconcert with 7000 cGy XRT results in a response lasting 10 mo. Thepatient is then started on Tarceva maintenance therapy, which hecontinues until he was considered for IMMU-132 (hRS7-CL2A-SN-38) trial,in addition to undergoing a lumbar laminectomy. He receives the firstdose of IMMU-132 after 5 months of Tarceva, presenting at the time witha 5.6-cm lesion in the right lung with abundant pleural effusion. Hecompletes his 6^(th) dose two months later when the first CT shows theprimary target lesion reduced to 3.2 cm. Periodic assays for Trop-2⁺CTCs show a substantial reduction in CTC number following treatment withIMMU-132.

This Example shows the feasibility of selecting for patients who areresponsive to therapy with IMMU-132 or another therapeutic anti-Trop-2antibody, by assaying for Trop-2⁺ CTCs in the individual patient's bloodand/or determinining Trop-2 copy number in CTCs. The Example furtherdemonstrates the feasibility of monitoring relative levels of Trop-2⁺CTCs as an indicator of the efficacy of anti-Trop-2 based therapies.Preferably, patients who show a positive response, including but notlimited to a complete response (CR), partial response (PR) and/or stabledisease (SD) will show a decrease in levels of Trop-2⁺ CTCs of at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98% or at least 99%. Where the treatment is highlyefficacious and results in complete response, a decrease of 100% inTrop-2⁺ CTCs may be observed.

Example 11. Isolation and Detection of Trop-2⁺ CTCs with a VerIFASTSystem

A VerIFAST system as disclosed in Casavant et al. (2013, Lab Chip13:391-6; 2014, Lab Chip 14:99-105) is used to detect Trop-2+ CTCs inTNBC. 7.5 ml blood samples are collected from a series of patients withsuspected TNBC or control normal individuals and stored in CellSavetubes (Veridex LLC). Biotinylated anti-Trop-2 hRS7 antibody is preparedas disclosed in Casavant et al. (2013, Lab Chip 13:391-6). The bloodsamples are mixed with biotinylated anti-Trop-2 antibody andstreptavidin-conjugated PMPs (Casavant et al., 2013, Lab Chip 13:391-6)and CTCs are separated with the VerIFAST platform and a handheld magnet.Cells are stained for tumor markers and cell nuclei are fluorescentlylabeled with DAPI nuclear dye. The results show the presence ofcirculating Trop-2⁺ tumor cells in blood samples from individuals withTNBC, but not control normal individuals.

Example 12. Clinical Trials With IMMU-132 Anti-Trop-2 ADC ComprisinghRS7 Antibody Conjugated to SN-38

Summary

The present Example reports results from a phase I clinical trial andongoing phase II extension with IMMU-132 (sacituzumab govitecan), anantibody-drug conjugate (ADC) of the internalizing, humanized, hRS7anti-Trop-2 antibody conjugated by a pH-sensitive linker to SN-38 (meandrug-antibody ratio=7.6). Trop-2 is a type I transmembrane,calcium-transducing, protein expressed at high density (˜1×10⁵),frequency, and specificity by many human carcinomas, with limited normaltissue expression. Preclinical studies in nude mice bearing Capan-1human pancreatic tumor xenografts have revealed IMMU-132 is capable ofdelivering as much as 136-fold more SN-38 to tumor than derived from amaximally tolerated irinotecan therapy (not shown).

The present Example reports the initial Phase I trial of 25 patients(pts) who had failed multiple prior therapies (some includingtopoisomerase-I/II inhibiting drugs), and the ongoing Phase II extensionnow reporting on 69 pts, including in colorectal (CRC), small-cell andnon-small cell lung (SCLC, NSCLC, respectively), triple-negative breast(TNBC), pancreatic (PDC), esophageal, and other cancers.

As discussed in detail below, Trop-2 was not detected in serum, but wasstrongly expressed (≥2⁺) in most archived tumors. In a 3+3 trial design,IMMU-132 was given on days 1 and 8 in repeated 21-day cycles, startingat 8 mg/kg/dose, then 12 and 18 mg/kg before dose-limiting neutropenia.To optimize cumulative treatment with minimal delays, phase II isfocusing on 8 and 10 mg/kg (n=30 and 14, respectively). In 49 ptsreporting related AE at this time, neutropenia ≥G3 occurred in 28% (4%G4). Most common non-hematological toxicities initially in these ptshave been fatigue (55%; ≥G3=9%), nausea (53%; ≥G3=0%), diarrhea (47%;≥G3=9%), alopecia (40%), and vomiting (32%; ≥G3=2%). Homozygous UGT1A1*28/*28 was found in 6 pts, 2 of whom had more severe hematological andGI toxicities. In the Phase I and the expansion phases, there are now 48pts (excluding PDC) who are assessable by RECIST/CT for best response.Seven (15%) of the patients had a partial response (PR), includingpatients with CRC (N=1), TNBC (N=2), SCLC (N=2), NSCLC (N=1), andesophageal cancers (N=1), and another 27 pts (56%) had stable disease(SD), for a total of 38 pts (79%) with disease response; 8 of 13CT-assessable PDC pts (62%) had SD, with a median time to progression(TTP) of 12.7 wks compared to 8.0 weeks in their last prior therapy. TheTTP for the remaining 48 pts is 12.6+ wks (range 6.0 to 51.4 wks).Plasma CEA and CA19-9 correlated with responses. No anti-hRS7 oranti-SN-38 antibodies were detected despite dosing over months. Theconjugate cleared from the serum within 3 days, consistent with in vivoanimal studies where 50% of the SN-38 was released daily, with >95% ofthe SN-38 in the serum being bound to the IgG in a non-glucoronidatedform, and at concentrations as much as 100-fold higher than SN-38reported in patients given irinotecan. These results show that thehRS7-SN-38-containing ADC is therapeutically active in metastatic solidcancers, with manageable diarrhea and neutropenia.

Pharmacokinetics

Two ELISA methods were used to measure the clearance of the IgG (capturewith anti-hRS7 idiotype antibody) and the intact conjugate (capture withanti-SN-38 IgG/probe with anti-hRS7 idiotype antibody). SN-38 wasmeasured by HPLC. Total IMMU-132 fraction (intact conjugate) clearedmore quickly than the IgG (not shown), reflecting known gradual releaseof SN-38 from the conjugate. HPLC determination of SN-38 (Unbound andTOTAL) showed >95% the SN-38 in the serum was bound to the IgG. Lowconcentrations of SN-38G suggest SN-38 bound to the IgG is protectedfrom glucoronidation. Comparison of ELISA for conjugate and SN-38 HPLCrevealed both overlap, suggesting the ELISA is a surrogate formonitoring SN-38 clearance.

A summary of the dosing regiment and patient poll is provided in Table6.

TABLE 6 Clinical Trial Parameters Dosing Once weekly for 2 weeksadministered every 21 days regimen for up to 8 cycles. In the initialenrollment, the planned dose was delayed and reduced if ≥G2treatment-related toxicity; protocol was amended to dose delay andreduction only in the event of ≥G3 toxicity. Dose level 8, 12, 18 mg/kg;later reduced to an intermediate cohorts dose level of 10 mg/kg. Cohortsize Standard Phase I [3 + 3] design; expansion includes 15 patients inselect cancers. DLT G4 ANC ≥ 7 d; ≥G3 febrile neutropenia of anyduration; G4 Plt ≥ 5 d; G4 Hgb; Grade 4 N/V/D any duration/GS N/V/Dfor >48 h; G3 infusion- related reactions; related ≥G3 non-hematologicaltoxicity. Maximum Maximum dose where ≥ 2/6 patients tolerate 1^(st) 21-dAcceptable cycle w/o delay or reduction or ≥G3 toxicity. Dose (MAD)Patients Metastatic colorectal, pancreas, gastric, esophageal, lung(NSCLC, SCLC), triple-negative breast (TNBC), prostate, ovarian, renal,urinary bladder, head/neck, hepatocellular. Refractory /relapsed afterstandard treatment regimens for metastatic cancer. Prioririnotecan-containing therapy NOT required for enrollment. No bulkylesion >5 cm. Must be 4 weeks beyond any major surgery, and 2 weeksbeyond radiation or chemotherapy regimen. Gilbert's disease or known CNSmetastatic disease are excluded.

Clinical Trial Status

A total of 69 patients (including 25 patients in Phase I) with diversemetastatic cancers having a median of 3 prior therapies were reported.Eight patients had clinical progression and withdrew before CTassessment. Thirteen CT-assessable pancreatic cancer patients wereseparately reported. The median TTP (time to progression) in PDCpatients was 11.9 wks (range 2 to 21.4 wks) compared to median 8 wks TTPfor the preceding last therapy.

A total of 48 patients with diverse cancers had at least 1 CT-assessmentfrom which Best Response (not shown) and Time to Progression (TTP; notshown) were determined. To summarize the Best Response data, of 8assessable patients with TNBC (triple-negative breast cancer), therewere 2 PR (partial response), 4 SD (stable disease) and 2 PD(progressive disease) for a total response [PR+SD] of 6/8 (75%). ForSCLC (small cell lung cancer), of 4 assessable patients there were 2 PR,0 SD and 2 PD for a total response of 2/4 (50%). For CRC (colorectalcancer), of 18 assessable patients there were 1 PR, 11 SD and 6 PD for atotal response of 12/18 (67%). For esophageal cancer, of 4 assessablepatients there were 1 PR, 2 SD and 1 PD for a total response of ¾ (75%).For NSCLC (non-small cell lung cancer), of 5 assessable patients therewere 1 PR, 3 SD and 1 PD for a total response of ⅘ (80%). Over allpatients treated, of 48 assessable patients there were 7 PR, 27 SD and14 PD for a total response of 34/48 (71%). These results demonstratethat the anti-TROP-2 ADC (hRS7-SN-38) showed significant clinicalefficacy against a wide range of solid tumors in human patients.

The reported side effects of therapy (adverse events) are summarized inTable 7. The therapeutic efficacy of hRS7-SN-38 was achieved at dosagesof ADC showing an acceptably low level of adverse side effects. Bycomparison, patients receiving a dosage of irinotecan (125 mg/m²weekly×4, Q6W) showed a much higher incidence of adverse effects, with38% incidence of grade ¾ diarrhea, 31% neutropenia and 8% neutropenicfever/infection.

TABLE 7 Related Adverse Events Listing for IMMU-132, Starting does of 8or 10 mg/kg Criteria: Grade 3-4 Adverse Event for >5% or any Grade 3 or4 Adverse Event (N = 123 patients) Grade 3 Grade 4 Neutropenia 22 (18%)7 (6%) Febrile Neutropenia 3 (2%) 2 (2%) Diarrhea 4 (3%) 0 Anemia 7 (6%)0 Fatigue 6 (5%) 0 Vomiting 2 (2%) 0 WBC Decrease 2 (2%) 0 LymphocyteDecrease 2 (2%) 0 Asthenia 1 (1%) 0 Dizziness 1 (1%) 0 Urinary TractInfection 1 (1%) 0 Alopecia — —

Data on dose reduction is also summarized. Of 76 patients starting at adose of 8 mg/kg, 12 (16%) were provided with a dose reduction. Of 33patients at a starting dose of 10 mg/kg, 5 (15%) were provided with adose reduction. Of 9 patients at a starting dose of 12 mg/kg, 6 (67%)were provided with a dose reduction. Of 3 patients at a starting dose of18 mg/kg, 3 (100%) were provided with a dose reduction. We conclude thatat 8 and 10 mg/kg, there were few dose reductions, reflecting a mild,predictable and manageable toxicity profile at therapeutic levels ofADC. Currently, 425 serum samples from 148 patients have been analyzedand no evidence of an antibody response to IMMU-132 has been detected,even after repeated administration, with some patients receiving morethan 20 doses of ADC.

Of 46 assessable patients with TNBC treated to date (Phase I and II), anobjective response was seen in 12 patients (26%), with disease controlin 34 patients (74%), a clinical benefit ratio (CR+PR+(SD≥6 mo)] of 46%and a clinical benefit ratio (CR+PR+(SD≥4 mo)] of 63%.

Of 19 assessable patients with NSCLC treated to date, an objectiveresponse was seen in 6 patients (32%), with disease control in 14patients (74%), and a clinical benefit ratio (CR+PR+(SD≥4 mo)] of 59%.

Of 20 assessable patients with SCLC treated to date, an objectiveresponse was seen in 6 patients (30%), with disease control in 11patients (55%), a clinical benefit ratio (CR+PR+(SD≥6 mo)] of 37% and aclinical benefit ratio (CR+PR+(SD≥4 mo)] of 55%.

Of 16 assessable patients with EAC treated to date, an objectiveresponse was seen in 2 patients (13%), with disease control in 9patients (56%), and a clinical benefit ratio (CR+PR+(SD≥4 mo)] of 44%.

Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CTdata (not shown). As an exemplary PR in CRC, a 62-year-old woman firstdiagnosed with CRC underwent a primary hemicolectomy. Four months later,she had a hepatic resection for liver metastases and received 7 mos oftreatment with FOLFOX and 1 mo 5FU. She presented with multiple lesionsprimarily in the liver (3+ Trop-2 by immunohistology), entering thehRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year afterinitial diagnosis. On her first CT assessment, a PR was achieved, with a37% reduction in target lesions (not shown). The patient continuedtreatment, achieving a maximum reduction of 65% decrease after 10 monthsof treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5ng/mL), before progressing 3 months later.

As an exemplary PR in NSCLC, a 65-year-old male was diagnosed with stageIIIB NSCLC (sq. cell). Initial treatment of caboplatin/etoposide (3 mo)in concert with 7000 cGy XRT resulted in a response lasting 10 mo. Hewas then started on Tarceva maintenance therapy, which he continueduntil he was considered for IMMU-132 trial, in addition to undergoing alumbar laminectomy. He received first dose of IMMU-132 after 5 months ofTarceva, presenting at the time with a 5.6 cm lesion in the right lungwith abundant pleural effusion. He had just completed his 6^(th) dosetwo months later when the first CT showed the primary target lesionreduced to 3.2 cm (not shown).

As an exemplary PR in SCLC, a 65-year-old woman was diagnosed withpoorly differentiated SCLC. After receiving carboplatin/etoposide(Topo-II inhibitor) that ended after 2 months with no response, followedwith topotecan (Topo-I inhibitor) that ended after 2 months, also withno response, she received local XRT (3000 cGy) that ended 1 month later.However, by the following month progression had continued. The patientstarted with IMMU-132 the next month (12 mg/kg; reduced to 6.8 mg/kg;Trop-2 expression 3+), and after two months of IMMU-132, a 38% reductionin target lesions, including a substantial reduction in the main lunglesion occurred (not shown). The patient progressed 3 months later afterreceiving 12 doses.

These results are significant in that they demonstrate that theanti-Trop-2 ADC was efficacious, even in patients who had failed orprogressed after multiple previous therapies.

In conclusion, at the dosages used, the primary toxicity was amanageable neutropenia, with few Grade 3 toxicities. IMMU-132 showedevidence of activity (PR and durable SD) in relapsed/refractory patientswith triple-negative breast cancer, small cell lung cancer, non-smallcell lung cancer, colorectal cancer and esophageal cancer, includingpatients with a previous history of relapsing on topoisomerase-Iinhibitor therapy. These results show efficacy of the anti-Trop-2 ADC ina wide range of cancers that are resistant to existing therapies.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

What is claimed is:
 1. A method of treating Trop-2⁺ tumors comprising:a) exposing an anti-Trop-2 antibody or antigen-binding fragment thereofto blood from a subject suspected of having a Trop-2⁺ cancer, whereinthe anti-Trop-2 antibody or fragment thereof is the sole anti-TAA (tumorassociated antigen) capture antibody; b) allowing the anti-Trop-2antibody or fragment thereof to bind to Trop-2⁺ circulating tumor cells(CTCs); and c) detecting CTCs bound to the anti-Trop-2 antibody orfragment thereof; and d) treating the subject with an anti-Trop-2antibody conjugated to at least one therapeutic agent.
 2. The method ofclaim 1, further comprising analyzing the copy number of Trop-2 in theCTCs.
 3. The method of claim 2, wherein the presence of Trop-2⁺ CTCswith a high copy number of Trop-2, wherein a high copy number is 3 ormore per cell, is predictive of response to a therapeutic anti-Trop-2antibody.
 4. The method of claim 1, further comprising monitoring thepresence to Trop-2⁺ CTCs in the circulation to determine the response ofthe tumor to therapeutic anti-Trop-2 antibody.
 5. The method of claim 1,wherein the therapeutic agent is selected from the group consisting ofan antibody, an antibody fragment, a drug, a toxin, a hormone, animmunomodulator, a pro-apoptotic agent, an anti-angiogenic agents, aboron compound, a photoactive agent and a radionuclide.
 6. The method ofclaim 5, wherein the drug is selected from the group consisting of ananthracycline, a camptothecin, a tubulin inhibitor, a maytansinoid, acalicheamycin, an auristatin, a nitrogen mustard, an ethyleniminederivative, an alkyl sulfonate, a nitrosourea, a triazene, a folic acidanalog, a taxane, a COX-2 inhibitor, a pyrimidine analog, a purineanalog, an antibiotic, an enzyme inhibitor, an epipodophyllotoxin, aplatinum coordination complex, a vinca alkaloid, a substituted urea, amethyl hydrazine derivative, an adrenocortical suppressant, a hormoneantagonist, an antimetabolite, an alkylating agent, an antimitotic, ananti-angiogenic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, aheat shock protein (HSP90) inhibitor, a proteosome inhibitor, an HDACinhibitor, and a pro-apoptotic agent.
 7. The method of claim 5, whereinthe drug is selected from the group consisting of 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil,cisplatinum, COX-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, DM1, DM3, DM4, doxorubicin, 2-pyrrolinodoxorubicine(2-PDox), a pro-drug form of 2-PDox (pro-2-PDox), cyano-morpholinodoxorubicin, doxorubicin glucuronide, endostatin, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,monomethylauristatin F (MMAF), monomethylauristatin D (MMAD),monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, SN-38, sorafenib,streptozocin, SU11248, sunitinib, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839.
 8. The method of claim 5,wherein the drug is selected from the group consisting of SN-38,pro-2-pyrrolinodoxorubicin (pro-2-PDox), paclitaxel, calichemicin, DM1,DM3, DM4, MMAE, MMAD and MMAF.
 9. The method of claim 1, wherein thecancer is resistant to treatment with at least one anti-cancer therapy.10. The method of claim 1, wherein the cancer is pancreatic cancer. 11.The method of claim 5, wherein the radionuclide is selected from thegroup consisting of ¹¹C, ¹³N, ¹⁵O, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁸Co, ⁵⁹Fe,⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br, ^(80m)Br,⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo, ^(99m)Tc, ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru,¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt, ¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te,^(122m)Te, ¹²⁵I, ^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm,¹⁵²Dy, ¹⁵³Sm, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er,¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt,¹⁹⁸Au, ¹⁹⁹Au, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb,²¹³Bi, ²¹⁵Po, ²¹⁷At, ²¹⁹Rn, ²²¹Fr, ²²³Ra, ²²⁴Ac, ²²⁵Ac, ²⁵⁵Fm and ²²⁷Th.12. The method of claim 5, wherein the toxin is selected from the groupconsisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase),DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonasendotoxin.
 13. The method of claim 5, wherein the immunomodulator isselected from the group consisting of a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), an interleukin, erythropoietin andthrombopoietin.
 14. The method of claim 13, wherein the cytokine isselected from the group consisting of human growth hormone, N-methionylhuman growth hormone, bovine growth hormone, parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, thrombopoietin (TPO),NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growthfactor-I, insulin-like growth factor-II, erythropoietin (EPO),osteoinductive factors, interferon-α, interferon-β, interferon-γ,macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin,endostatin, tumor necrosis factor and lymphotoxin.
 15. The method ofclaim 1, wherein the anti-Trop-2 antibody or fragment thereof binds tothe same epitope as an anti-Trop-2 antibody comprising the light chainCDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ IDNO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).
 16. The method of claim 1,wherein the anti-Trop-2 antibody or fragment thereof is an RS7 antibodycomprising light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1);CDR2 (SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and theheavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6).